The purpose of this research is to provide new information which will lead to a better understanding of the mechanisms which regulate the concentrations of glutamate, GABA and aspartate within specific compartments (cellular, sub-cellular and extracelluar) of the CNS. Our approach is to investigate the uptake and metabolism of these amino acids and metabolically related compounds by enriched and pure populations of cells and sub-cellular particles (e.g., synaptosomes). The effects of various potential regulators (e.g., cyclic nucleotides, N-acetylaspartate and N-acetylglutamate) on uptake and metabolism is being examined. Methodologies needed to obtain specific types of cells are being developed and employed to isolate specific types of neurons and glial cells. These include the generation of monoclonal antibodies to membrane antigens of specific types of cells, and subsequent conjugation of these antibodies to magnetic microspheres. The use of a magnetic field can then be used to separate one population of cells from another. The uptake and metabolism of amino acid neurotransmitters by cells grown in vitro is also being studied in order to determine changes in neurotransmitter metabolism as a function of cell differentiation. Recently we have studied the localization of pyruvate carboxylase using immunocytochemical and biochemical assay procedures. Our research indicates that this enzyme is astrocyte specific in CNS tissues. This observation provides further evidence for our working hypothesis that a-ketoglutarate is synthesized in astrocytes then made available to nerve terminals where it is used to replenish the neurotransmitter pools of glutamate and GABA.